Mammalian cells have multiple mechanisms for regulating chromatin structure and dynamics. These mechanisms include enzymatic modification of nucleosomes, core histone variants, tethering of chromosomal sites via chromatin modifying enzymes, chromatin remodelers, chromatin architectural proteins (CAPs), and a panoply of RNAs. Abnormalities of nucleome architecture have been implicated in diseases including Down's Syndrome, metabolic diseases, neurological disorders such as Rett syndrome, and cancers of the breast, lung, and brain. Unfortunately, it has proven difficult to relate local biochemical modifications within the chromatin to the broader organization, dynamics, and function of the nucleome. The difficulty of making these connections reflects the complexity of the underlying biology and several critical gaps in our measurement capabilities. The nucleome research community currently has (1) tools with whole-genome coverage but with limited cell-to-cell and temporal resolution and (2) tools with high spatial and temporal resolutio but with limited coverage, short measurement duration, and poor performance in the `thick' cells that constitute most of the cell types in the human body. We are a team of investigators from the areas of super-localization microscopy, optical labels, stem cell and cancer biology, and chromatin theory. Together, we have developed a pair of connected technologies for 4D nucleomics. We propose to now take the next step, and refine and stringently test: (1) a new class of genetically encoded and effectively unbleachable fiducials for multiplexed labeling of chromatin components, and (2) a 4D super-localization microscope for simultaneously tracking many targets within `thick' (~10 micron diameter) nuclei. With the labels, the imaging of single molecules within living nuclei is no longer temporally limited by photo bleaching, a new capability in light microscopy. The hardware is designed for simultaneous 4D super- localization tracking of many loci in cell types such as human stem cells, a key hardware capability for nucleome biology. To facilitate the spread of the tools into the broader biological community, we will test our tools in two systems, (1) the role of the USP16 deubiquitinase enzyme in human Down's Syndrome and (2) alterations of loci dynamics in the presence of the SATB1 DNA `loopscape controller'. When combined and validated as a unit, the tools are designed to allow measurement of the nucleome's architecture and dynamics during major cellular transitions such as division, differentiation, and senescence.